Antibiofilm Peptides Increase the Susceptibility of Carbapenemase-Producing Klebsiella pneumoniae Clinical Isolates to β-Lactam Antibiotics

Abstract

dMultidrug-resistant carbapenemase-producing Klebsiella pneumoniae (KpC) strains are becoming a common cause of infections in health care centers. Furthermore, Klebsiella can develop multicellular biofilms, which lead to elevated adaptive antibiotic resistance. Here, we describe the antimicrobial and antibiofilm activities of synthetic peptides DJK-5, DJK-6, and 1018 against five KpC isolates. Using static microplate assays, it was observed that the concentration required to prevent biofilm formation by these clinical isolates was below the MIC for planktonic cells. More-sophisticated flow cell experiments confirmed the antibiofilm activity of the peptides against 2-day-old biofilms of different KpC isolates, and in some cases, the peptides induced significant biofilm cell death. Clinically relevant combinations of DJK-6 and ␤-lactam antibiotics, including the carbapenem meropenem, also prevented planktonic growth and biofilm formation of KpC strain1825971. Interestingly, peptide DJK-6 was able to enhance, at least 16-fold, the ability of meropenem to eradicate preformed biofilms formed by this strain. Using peptide DJK-6 to potentiate the activity of ␤-lactams, including meropenem, represents a promising strategy to treat infections caused by KpC isolates. Klebsiella pneumoniae is a Gram-negative, encapsulated, opportunistic pathogen of the family Enterobacteriaceae (1). It has become a significant cause of infections, especially pneumonia, but also meningitis, sepsis, and urinary tract, device-associated, and surgical wound site infections (2-7). The most severe clinical cases of such infections are often associated with K. pneumoniae strains that produce a plasmid-borne carbapenemase (KpC).The rate of fatality from KpC-producing K. pneumoniae is high (Ͼ50%) among patients with bloodstream infection (2,8,9).The treatment of these infections is difficult since KpC strains are resistant to all ␤-lactam antibiotics, including carbapenems, which are typically used as a last resort (10). In addition, bacteria harboring carbapenemase genes are often resistant to other antibiotics. As a consequence of this multidrug resistance, failure in treatment contributes to increased rates of morbidity and mortality (9,11,12). Tigecycline, aminoglycosides, and the polymyxins (polymyxin B and colistin) are currently the best treatment options for carbapenemase-producing strains (13). However, these strains also have the potential to develop resistance to these antibiotics (14-16).The treatment of infections caused by multidrug-resistant bacteria is a challenge, and the ability of bacterial pathogens to form biofilms can further complicate this scenario. Biofilms, a distinct microbial lifestyle of bacteria, are surface-associated multicellular communities of bacteria encased in a protective polymeric matrix that can include polysaccharides, proteins, and DNA (17). They represent a substantial problem in the clinic, as they can lead to the development of chronic long-lived infections on body surfaces, including the lung, skin, hea...

Synthetic antibiofilm peptides

“…Several other combinations of DJK-5 and DLK-6 with different antibiotics led to the eradication of P. aeruginosa , E. coli , A. baumannii , K. pneumoniae and S. enterica [18]. Intriguingly, Ribeiro et al demonstrated similar findings, reporting that peptide DJK-6 enhanced the activity of the β-lactam antibiotics meropenem, imipenem and cefepime to prevent biofilm formation by carbapenemase KpC-producing clinical isolates [39]. The combination of this peptide at 0.125 μg.mL −1 with only 0.06 μg.mL −1 of meropenem was capable of eradicating mature biofilms formed by these KpC isolates.…”

“…Importantly, the peptides were shown to be non-toxic in the same invertebrate studies. In a parallel study performed by Ribeiro et al [39], it was demonstrated that DJK-6 was a promising peptide in preventing the formation of biofilms, as well as eradicating preexisting biofilms (at 2 – 4 μg.mL −1 ) formed by carbapenemase-producing K. pneumoniae clinical isolates (KpC isolates) [39]. …”

“…The combination of this peptide at 0.125 μg.mL −1 with only 0.06 μg.mL −1 of meropenem was capable of eradicating mature biofilms formed by these KpC isolates. This represents at least a 16-fold decrease in the concentration of antibiotic required to eradicate such biofilms [39]. It would be interesting to determine whether peptides such as DJK-6 act as β-lactamase inhibitors, as this has been shown to be an activity of certain peptides [41].…”

An Immunomodulatory Peptide Confers Protection in an Experimental Candidemia Murine Model

“…Biofilm formation by the C. albicans CI was assessed in BM2 minimal medium, consisting of 62 mM potassium phosphate buffer (pH 7.0), 7 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 10 M FeSO 4 , and 0.5% glucose. The minimal biofilm inhibitory concentration to inhibit biofilm formation by 50% (MBIC 50 ) or 100% (MBIC 100 ) was assessed as described previously by Ribeiro et al (38). Briefly, after removal of planktonic cells, residual adherent cells were stained with 0.01% crystal violet for 20 min, and deionized water was used to wash microplate wells twice.…”

Antibiofilm Peptides: Potential as Broad-Spectrum Agents

“…Thus, these peptides represent a genuine antiresistance strategy for biofilm bacteria. Indeed, antibiofilm peptides were recently shown to reverse K. pneumoniae carbapenemase (KPC)-mediated resistance in K. pneumoniae, in that, e.g., 0.0625 g/ml meropenem combined with 0.125 g/ml DJK5 led to complete eradication of preexisting flow cell biofilms (40). …”